Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    with heavy donor DNA and then hybrid density DNA with transforming activity was reextracted after varying times. This DNA was then dena tured into its single strands and annealed with wild type (light density) DNA lacking the transforming marker. The duplexes generated by this treatment were tested for their possession of the transforming marker. Since one strand of the reconstituted duplex does not carry the transform ing marker, its activity in transformation measures the presence of this marker on the strand that was annealed with it, that is the strand derived from the DNA extracted from transformed cells at different times. Thus any transforming activity in DNA of hybrid density is due to an original heavy donor strand; any activity in DNA of light density is due to a complement that must have been synthesized after integration of the donor material. According to this assay, at 10 minutes after addition of donor DNA almost all the transforming activity resides in the heavy strand; but at 40 minutes or 70 minutes, after one or two rounds of replication respectively, 50% of the activity was in the heavy and 50% in the light (newly synthesized) strands. The implication is that only one strand of transforming DNA need carry the donor information; and this may be either strand. A search for single stranded regions in donor DNA at various times after uptake by H.influenzae recipients was made by Sedgwick and Set- low (1976), taking advantage of the SI nuclease; this enzyme is extremely specific for single stranded DNA, removing any such regions from duplex DNA. In these experiments competent cells were incubated with labeled transforming DNA and uptake then terminated by addition of DNAase. Immediately following this protocol, about 13% of the donor DNA taken up by the cells was degraded by SI nuclease; and over the next 40 minutes this value declined to about 4%. When the rec2 or KBÌ mutants of H.influenzae which appear to be deficient in the ability to integrate donor DNA are used as recipients, sensitivity to SI nuclease does not appear in the donor DNA. This suggests that the generation of single stranded regions in the donor material is necessary for transformation. What is the location of the single stranded regions in the donor molecules? Because the size of the donor DNA appears to be unchanged by treatment with SI nuclease, as judged by its rate of sedimentation through sucrose gra dients, the single stranded regions may lie at the ends of the donor molecules (rather than within where their cleavage would reduce the size). During acquisition of competence in H.influenzae single stranded re gions appear in the recipient genome. LeClerc and Setlow (1975) found that when the DNA of competent cells is sedimented through alkaline sucrose gradients, a pulse label given after the acquisition of competence